Ribosome Display Technology Research Articles

Development of uniform ribosome display technology enabling easy and efficient identification of full-length proteins that interact with bioactive small and large molecules.

Identifying target proteins that interact with bioactive molecules is indispensable for understanding their mechanisms of action. In this study, we developed a uniform ribosome display technology using equal-length DNAs and mRNAs to improve molecular display principle for target identification. The equal-length DNAs were designed to contain various coding sequences for full-length proteins with molecular weights of up to 130 kDa and were used to synthesize equal-length mRNAs, which allowed the formation of full-length protein-ribosome-equal-length mRNA complexes. Uniform ribosome display selections of dihydrofolate reductase and haloalkane dehalogenase mutant were performed against methotrexate and chlorohexane, respectively. Quantitative changes of proteins after each selection indicated that the target protein-displaying ribosomal complexes were specifically selected through non-covalent or covalent interactions with the corresponding bioactive molecules. Furthermore, selection of full-length proteins interacting with methotrexate or anti-DDX46 antibody from protein pools showed that only the target proteins could be precisely identified even though the molar amounts of equal-length mRNAs encoding them were adjusted to 1/20,000 of the total equal-length mRNAs. Thus, the uniform ribosome display technology enabled efficient identification of target proteins that interact with bioactive small and large molecules through simplified operations without deep sequencing.

Antiviral Drugs and Vaccines for Omicron Variant: A Focused Review.

The Omicron variant of concern (VOC) replaced the delta variant rapidly and became the predominant strain due to more mutations in spike protein and receptor-binding domain (RBD) enhancing its infectivity and binding affinity. The severity of the illness is less than that of the delta variant. Omicron is nonsusceptible to REGEN-COV™ and bamlanivimab with etesevimab. Drugs that are effective against the Omicron variant are oral antiviral drugs such as Paxlovid (nirmatrelvir/ritonavir), remdesivir, sotrovimab, and molnupiravir. The potency of sotrovimab is reduced to 3-fold against Omicron, and 8-fold reduction in potency with sotrovimab is found in a particular variant of Omicron with a R346K substitution in spike protein. There are neither clinical trials comparing the efficacy of these 4 therapies with each other nor any data on a combination of two or more therapies. The current recommendation for mild-moderate, nonhospitalized patients who are at a high risk of disease progression is to use Paxlovid as the first-line option. If Paxlovid is not available or cannot be administered due to drug interactions, then the next best choice is sotrovimab. The third choice is remdesivir if sotrovimab is also not available and molnupiravir is to be given if the other three options are not available or cannot be administered. For prevention, 2130 (cilgavimab) in combination with COV2-2196 (tixagevimab) has been effective against BA.2 only. LY-CoV1404 (bebtelovimab) is recently authorized as it is effective against all sublineages of the Omicron variant. Regarding vaccine efficacy (VE), the 3-dose VE with mRNA vaccines at 14-60 days was found to be 71.6%, and after 60 days, it is 47.4%. There is a 34-38-fold reduction of neutralizing activity with prebooster sera and a 19-fold reduction with booster sera for the Omicron variant. This probably explains the reason for worldwide breakthrough infections with the Omicron variant with waning immunity. The neutralizing antibody response against Omicron elicited by the bivalent vaccine is superior to that of the ancestral Wuhan strain, without any safety concerns. For future advances, the ribosome display technology can be applied for the generation of human single-chain fragment variable (scFv) antibodies from B cells of recovered patients against Omicron and other Coronavirus variants as they are easier and faster to produce and have high affinity and high specificity.

Retrospective analysis of the preparation and application of immunotherapy in cancer treatment (Review).

Monoclonal antibody technology plays a vital role in biomedical and immunotherapy, which greatly promotes the study of the structure and function of genes and proteins. To date, monoclonal antibodies have gone through four stages: murine monoclonal antibody, chimeric monoclonal antibody, humanised monoclonal antibody and fully human monoclonal antibody; thousands of monoclonal antibodies have been used in the fields of biology and medicine, playing a special role in the pathogenesis, diagnosis and treatment of disease. In this review, we compare the advantages and disadvantages of hybridoma technology, phage display technology, ribosome display technology, transgenic mouse technology, single B cell monoclonal antibody generation technologies, and forecast the promising applications of these technologies in clinical medicine, disease diagnosis and tumour treatment.

Generating highly potent and efficacious antibodies to the cholecystokinin B (CCK‐B) receptor by ribosome display for the treatment of neuropathic pain

Development of effective non‐opioid therapeutics tailored to specific chronic pain syndromes is sorely needed. Our recent microchip gene expression profile identified upregulated cholecystokinin B (CCK‐B) receptor in trigeminal ganglia (TG) as one suitable target among many others. Thus, the CCK‐B receptor is an ideal candidate to impact both nociceptive and limbic components of chronic pain. More importantly, its inhibition can reverse opiate tolerance. Based on the literature and the gene expression changes over time, the CCK‐B receptor is implicated as one of the persisting functional targets suitable for development of therapeutics to inhibit chronic pain‐ and anxiety‐related behaviors. Here we describe the isolation and characterization of a panel of CCK‐B receptor specific antibodies using ribosome display technology. Furthermore, we demonstrate in vivo efficacy of our antibodies in a relevant model of chronic neuropathic pain. This is the first example, to date, of scFv antibodies that can block the CCK‐B receptor directly with high potency and specificity. This highlights the exciting potential therapeutic opportunity of an antibody for blocking this type of receptor.Support or Funding InformationThis work was supported by Startup dollars from Loyola University Medical Center (RD) and University of New Mexico (KW).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Screening and application of peptamers

Biosensors have undergone considerable development in recent years, and research focused on aptamers has played an important role in promoting the development of biosensors. Peptamers are short peptides that can specifically bind to target substances. The development of peptamers has been limited by screening, synthesis, and purification methods; however, their high affinity, strong specificity, and good biological compatibility combined with the development of new simulation tools for peptamer screening methods reveal their many potential applications. Peptamer screening may be done in a number of ways; in addition to the traditional yeast two-hybrid technology, phage display, ribosome display technology, and molecular docking based on bioinformatics are accelerating the development of peptamers. Peptamers can be used not only in clinical medicine but also in the fields of precision nutrition, environmental monitoring, and new material identification. The purpose of this paper was to review the screening and applications of peptamers.

A novel bispecific DARPin targeting FcγRIIB and FcεRI-bound IgE inhibits allergic responses.

Binding of allergen-specific IgE to its high-affinity receptor FcεRI on basophils and mast cells is a central event in the development of allergies. Exposure of these cells to allergens induces the release of soluble mediators causing allergic symptoms. The inhibitory low-affinity IgG Fc-receptor FcγRIIB is co-expressed on allergic effector cells and has been implicated in negative regulation of immediate hypersensitivity responses. In order to harvest the inhibitory function of this receptor, we aimed to select specific binders against FcγRIIB and to generate a bispecific molecule simultaneously targeting FcγRIIB and FcεRI-bound IgE on the surface of allergic effector cells. We selected FcγRIIB-specific binding molecules from a library of designed ankyrin repeat proteins using ribosome display technology. The bispecific binding modality was generated by molecular cloning and recombinant protein expression. We determined binding characteristics on molecular and cellular levels using SPR, ELISA, and flow cytometry. The inhibitory potential of the newly described molecules was assessed in different cellular degranulation assays ex vivo and in a mouse model of passive systemic anaphylaxis. We demonstrate that the selected DARPin® proteins recognize FcγRIIB with high affinity. Furthermore, the bispecific binding protein successfully interferes with allergen-induced cell degranulation and efficiently inhibits systemic anaphylaxis in vivo. Mechanistically, we report that FcγRIIB-mediated inhibition of effector cell activation requires direct ligation to an activating FcεRI receptor. The described bispecific DARPin protein has the ability to co-ligate FcγRIIB with FcεRI-bound IgE on allergic effector cells and represents an efficient dual-modality to interfere with allergic hypersensitivity reactions.

Inhibitory effect of humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma: in vitro and in vivo studies

ObjectiveTo investigate the inhibitory effect of humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma both in vitro and in vivo, which may be a potential agents with sensitivity and targeting ability for human hepatocellular cancer. MethodsHumanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate was previously constructed using ribosome display technology and antibody conjugate technology. In this combined in vitro and in vivo study, the inhibitory effects of anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on tumor growth, invasion, and metastasis was observed with human liver carcinoma cell line Bel7402 and normal cell L02 by MTT assay, Tanswell assay, Hochest33258 staining, and DNA ladder analysis. The anticancer activity and distribution of anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles was then verified in a mouse model of Bel7402 xenografts. ResultsAnti-VEGFR-2 ScFv-As2O3-stealth nanoparticles significantly inhibited the proliferation of Bel7402 in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while had almost no effects on L02 cells. And the apoptosis inducing effects were proved by Hochest33258 staining and DNA ladder analysis. Transwell assay found that the drug also inhibited the metastasis ability of tumor cells. Furthermore, anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles significantly delayed the growth of Bel7402 xenografts after administration (92.9%), followed by As2O3-stealth nanoparticles, anti-VEGFR-2 ScFv, and As2O3 (61.4%, 58.8%, 20.5%, P<0.05). The concentration of As2O3 in anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles group was more selectively. ConclusionsAnti-VEGFR-2 ScFv-As2O3-stealth nanoparticles is a potent and selective anti-hepatocellular carcinoma agent which could inhibit the growth of liver cancer as a targeting agent both in vitro and in vivo and also significantly inhibit angiogenesis.

Humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles, an antibody conjugate with potent and selective anti-hepatocellular carcinoma activity

Low sensitivity of tumor tissue, targeting and sustained release of the drug are bottlenecks of the effect of chemotherapy on hepatocellular carcinoma. In this study, we used the ribosome display technology to screen human anti-VEGFR 2-single-chain antibody (ScFv) that could target directly to VEGFR2, and nanotechnology to prepare As2O3-nanoparticles. Then we built anti-VEGFR-2ScFv-As2O3-stealth nanoparticles using molecular coupling technology, which significantly increased anti-tumor effect while reducing toxicity. The in vivo tissue targeting distribution and anti-tumor effects of the anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles were investigated. Our results showed that anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles could inhibit the development of liver cancer xenograft as a targeting agent and also significantly inhibit angiogenesis.

Selection of bisphenol A – single-chain antibodies from a non-immunized mouse library by ribosome display

Developing reagents with high affinity and specificity are critical to detect the environmental hormones or toxicants. Ribosome display technology has been widely used in functional protein or peptide screening and in directed evolution of protein molecules in vitro. In this study, single-chain variable fragments (scFvs) against bisphenol A (BPA) were selected from a library constructed from splenocytes of non-immunized mice. After five rounds of selection, the selected scFvs bound to BPA with high affinity. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was introduced to screen the antibody affinity and specificity to BPA. The equilibrium dissociation constants (KDS) of one clone was 1.76μM as determined by surface plasmon resonance (SPR). This study indicated that ribosome display can isolate binders to small molecules from a non-immunized naive library without any in vivo steps and can generate recombinant antibodies efficiently and rapidly. In addition, this study provides a methodological framework for detection of small molecules using recombinant antibodies.

Preparation of Human Antibody Fragments Against Rabies Virus Based on Ribosome Display Technology*

To prepare human antibody to rabies virus with high neutralizing potency using ribosome display technology.The immunoglobulin heavy and light chain variable(VH,VL) genes were prepared with the peripheral blood lymphocytes from three volunteers immunized with rabies virus vaccine by PCR.The genes encoding scFv fragments were prepared by randomly combining VH and VL genes by SOE PCR.Rabies virus glycoprotein(RVGp) specific scFv genes were selected over five cycles of ribosome display.The isolated scFv genes were cloned into pET22b(+)/BL21(DE3),from which soluble scFv fragments were prepared.The expressed products of selected clones were analyzed by ELISA for isolating the positive clones.To improve the stability of obtained scFvs,VH-Lc-VK were constructed and characterized.A human scFv gene library with 6.2×1012 numbers used for ribosome display was constructed.Among the 180 selected clones,four clones RB24,RB71,RB109 and RB156 which exhibited the highest ELISA signals were isolated.The analysis of their sequences showed that they were new human immunoglobulin V genes to RVGp.And the reconstructed VH-Lc-VK antibody fragments can recognize RVGp specifically and antagonize the cytolytic effect of rabies virus on Vero cells.The prepared VH-Lc-VK fragments to RVGp will be useful for preparing engineering antibodies with high affinity against rabies virus.Ribosome display is a rapid means of generating fully human antibody fragments in vitro.

Selection and affinity maturation of human antibodies against rabies virus from a scFv gene library using ribosome display

We selected useful antibody fragments against rabies virus from a human single chain variable fragment (scFv) gene library using ribosome display technique. The recombinant rabies virus glycoprotein (RVGp) was used as an antigen to isolate specific scFvs. After five rounds of selection, the analysis demonstrated that scFv–ribosome–mRNA complexes were specifically selected against RVGp. Sequence analysis showed that mutations were introduced at random by PCR among the rounds of selection and variants with high affinity were isolated. The obtained scFvs with high affinity could recognize RVGp specifically and showed binding activity to rabies virus. These scFvs were potential for inclusion in a combination of several human monoclonal antibodies (MAbs) aimed for application in rabies post-exposure prophylaxis. Ribosome display technology is a robust tool for rapid isolation of human antibody fragments, and has exceptional strength in affinity maturation and molecular evolution in vitro.

Selection of scFvs specific for the HepG2 cell line using ribosome display

The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.

Ribosome display and selection of human anti-CD22 scFvs derived from an acute lymphocytic leukemia patient

Novel in vitro methods for the display of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) to isolate specific binding reagents to these antigens. Here, we present the isolation of human scFv against the immunotherapeutic target antigen CD22 from a patient-derived human scFv library using ribosome display technology. The ribosome complexes were enriched against the extra-cellular domain of human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific binding activity was demonstrated by surface plasmon resonance and validated using in vitro cell assays. The isolated human anti-CD22 scFvs can provide a basis for the development of new immunotherapeutic strategies in CD22-expressing malignant diseases.

Selection of Human Anti-CD22 scFvs from an Acute Lymphocytic Leukemia Related scFv Library with Ribosome Display.

Novel in vitro methods for screening of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Ribosome display represents an emerging and innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) in order to isolate specific binding reagents. Here we present the isolation of human scFv against the validated immunotherapeutic target antigen CD22 from a leukaemia patient-derived human scFv library using ribosome display technology. The ribosome-displayed scFv were enriched against the human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific target recognition was demonstrated by surface plasmon resonance and validated using in vitro cell assays.

10th Annual Drug Discovery Technology® Europe And Infotechpharma® Conference

The 10th Annual Drug Discovery Technology® Europe and InfoTechPharma® conference and exhibition were held on 14 – 16 March 2006 at the Novotel in London. The conference saw > 110 international speakers contribute their views on the latest topics in drug discovery, from novel experimental methods to future business models. This meeting offered an excellent platform for scientists and pharma IT specialists working along the drug discovery pipeline to debate the merits of various research strategies and exchange cutting edge ideas and R&D solutions. Selected presentations from the conference are further discussed in this meeting report, which covers ribosome display technology, translational medicine, research strategies for cardiovascular diseases and computer-aided molecular design.

An improved method for an efficient and easily accessible eukaryotic ribosome display technology

Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. However, this technology has so far been perceived as being technically challenging owing to comparatively difficult protocols and the absence of tailored commercial reagents, particularly when using prokaryotic cell-free expression systems. Eukaryotic ribosome display is potentially a more accessible alternative because of the availability of suitable commercial reagents, yet despite published protocols, this method has been less widely used. For eukaryotic ribosome display, a novel mechanism of mRNA recovery compared with that of the well-proven prokaryotic method has been proposed. We have examined the eukaryotic ribosome display process with the aims of investigating the proposed mechanism of sequence recovery and of identifying aspects of the protocol that may have lead to poor performance and therefore so far limited its use. We demonstrate that the proposed novel method is in fact mechanistically comparable to the prokaryotic method and we provide a step-by-step protocol for eukaryotic ribosome display that is 20-fold more efficient than current published methods. Our findings should increase the ease of operating ribosome display technology, making it more accessible to the scientific community.

Identification of a Functional Epitope of the Nogo Receptor by a Combinatorial Approach Using Ribosome Display

The Nogo receptor (NgR) plays a central role in mediating growth-inhibitory activities of myelin-derived proteins, thereby severely limiting axonal regeneration after injury of the adult mammalian central nervous system (CNS). The inhibitory proteins Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) all bind to the extracellular leucine-rich repeat (LRR) domain of NgR, which provides a large molecular surface for protein-protein interactions. However, epitopes within the LRR domain of NgR for binding Nogo, MAG and OMgp have not yet been revealed. Here, we report an evolutionary approach based on the ribosome display technology for detecting regions involved in ligand binding. By applying this method of "affinity fingerprinting" to the NgR ligand binding domain we were able to detect a distinct region important for binding to Nogo. Several residues defining the structural epitope of NgR involved in interaction with Nogo were subsequently confirmed by alanine scanning mutagenesis.

Ribosome display of mammalian receptor domains

Many mammalian receptor domains, among them a large number of potential therapeutic target proteins, are highly aggregation-prone upon heterologous expression in bacteria. This severely limits functional studies of such receptor domains and also their engineering towards improved properties. One of these proteins is the Nogoreceptor, which plays a central role in mediating the inhibition of axon growth and functional recovery after injury of the adult mammalian central nervous system. We show here that the ligand binding domain of the Nogoreceptor folds to an active conformation in ternary ribosomal complexes, as formed in ribosome display. In these complexes the receptor is still connected, via a C-terminal tether, to the peptidyl tRNA in the ribosome and the mRNA also stays connected. The ribosome prevents aggregation of the protein, which aggregates as soon as the release from the ribosome is triggered. In contrast, no active receptor was observed in phage display, where aggregation appears to prevent incorporation of the protein into the phage coat. This strategy sets the stage for rapidly studying defined mutations of such aggregation-prone receptors in vitro and to improve their properties by in vitro evolution using the ribosome display technology.

Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library

Picloram-specific variable fragments (V HHs) of heavy chain antibodies (HCAbs) were selected from a naı̈ve-llama library using ribosome display technology. A cDNA library of V HHs was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different V HHs all belonging to the V HH subfamily 1 were isolated. Following another three cycles of selection, only two of the seven V HHs persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured K Ds were 3 and 254 μM, respectively. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naı̈ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome display technology can compensate for the limited diversity of a V HH naı̈ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.